Bone marrow-derived cells for cardiovascular cell therapy: an optimized GMP method based on low-density gradient improves cell purity and function

Cardiovascular cell therapy represents a promising field, with several approaches currently being tested for the treatment of both heart disease and peripheral vascular diseases [1]-[3]. According to current European regulations [4]-[6], cell-based products such as bone marrow (BM)-derived cells for cardiovascular applications are defined as advanced therapy medicinal products (ATMP) and must be prepared according to Good Manufacturing Practice (GMP) standards. In this context, the development and validation of properly designed cell manufacturing and testing methods [7],[8] are of paramount importance for successful translational research. The manufacturing process has to be carefully defined and validated to ensure product consistency [7]. A suitable Quality Control (QC) strategy has to be designed for every specific ATMP, aiming at evaluating its safety, identity, purity and potency [7]-[9]. Safety testing should encompass sterility and lack of endotoxin, at least. The identity test panel includes cell morphology and immunophenotype. These tests also provide information on ATMP purity, as they detect undesirable impurities such as contaminating cell types. Potency is defined as a measure of biological activity. A potency assay should be based on a defined biological effect closely related to the mechanism(s) responsible for the functional benefits [7]. Cell viability is an important component of the potency of cell-based ATMP; however, additional parameters of biological activity should also be tested [10].

Release specifications (i.e. acceptance criteria to be met by a product lot in order to be administered to a patient) need to be defined for safety, which is generally evaluated by assays described in European Pharmacopoeia (EP) (compendial assays), and for crucial parameters such as cell viability. For other parameters, mostly evaluated by non-compendial assays developed on a product-specific basis, data may be recorded for information only, at least during the initial phases of clinical development.

Our Cell Therapy Unit, authorized since 2008 for the production of ATMP, is focused on development activities aimed at modifying research grade cell products to obtain high quality, clinical grade cell products.

The ATMP for the ongoing METHOD study ("Bone marrow derived cell therapy in the stable phase of chronic ischemic heart disease") [11] (ClinicalTrials.gov Identifier: NCT01666132; initial feasibility phase: 8/10 patients treated) consists of fresh mononuclear cells (MNC) isolated from autologous BM by density gradient centrifugation; cells are formulated in 5% human serum albumin (HSA) and tested for safety (sterility, endotoxin), identity/potency (cell count, CD45/CD34/CD133, viability) and purity (evaluation of contaminant granulocytes (GRA) and platelets (PLT).

The aims of the present work were (1) to optimize the manufacturing process in order to reduce contaminants; and (2) to set-up additional identity and potency assays in order to improve product characterization and evaluate product stability.

These improvements were crucial to the upcoming second phase of the METHOD trial, as well as to planned clinical trials, such as the CIRCULATE study ("Bone Marrow Derived Cell Therapy in Peripheral Artery Disease") in patients with critical limb ischemia. They have been explicitly requested by Swiss regulatory authorities (Swissmedic).

The BM-MNC manufacturing method currently used in the ongoing feasibility phase of the METHOD trial [11] is based on density gradient centrifugation (719 × g, 20' at 20°C) on standard Ficoll-Paque (1.077 g/L), followed by 3 washing steps (582 × g, 10' at 4°C).

The resulting product is composed of WBC with morphological characteristics of LYM (mean value ± standard deviation (SD)), 53% ± 16%), MON (15% ± 4%), and GRA (32% ± 18%) (n = 8). The PLT/WBC ratio is 4 ± 2. The MNC fraction comprising LYM and MON is considered the active fraction, whereas GRA and PLT represent impurities. For all 8 clinical lots produced so far, the levels of impurities were in compliance with product release specifications (GRA ≤ 75%, PLT/WBC ≤ 40; see below). Nonetheless, minimization of such impurities is desirable. Thus, development experiments were initiated to optimize the BM-MNC manufacturing process.

Preliminary tests performed on a limited number of samples indicated that gradient centrifugation parameters (relative centrifugation force and duration) may not affect process performance in terms of impurity clearance or cell yield (Table 1); however, changing the gradient medium may improve GRA removal (Table 2), while adding a low-speed washing step may improve PLT removal efficiency, with no negative impact on product potency (Table 3).

Based on such initial results, a new manufacturing method was designed, relying on the use of low-density Ficoll-Paque (1.073 g/L) as the gradient medium. The gradient step (400 × g, 30' at 20°C) was followed by 2 washing steps at 20°C, of which the second one was performed at low speed (washing #1: 400 × g; #2: 100 × g) in order to reduce the possibility that PLT may sediment together with MNC and be recovered in the cell pellet.

The new manufacturing method was compared with the previous one in terms of overall efficiency (Table 4). It resulted in significantly improved removal of contaminant PLT and GRA (p < 0.01). On the other hand, the MNC yield was diminished.

This work focused on the development and validation of a novel GMP manufacturing and testing strategy for BM-MNC as ATMP for cardiovascular cell therapy. Our goals were (1) to optimize the process in order to reduce contaminant GRA and PLT, and (2) to set-up additional identity and potency assays in order to better characterize the cell product and evaluate its stability.

In our product, the MNC fraction comprising LYM and MON is considered the active component, whereas PLT and GRA represent impurities. The role of PLT within a cell therapy product is controversial, presumably due to their complex biology: they may exert potential beneficial effects on cell engraftment and tissue regeneration [24], but also negative effects. For example, in the context of the REPAIR-AMI trial, the number of contaminating PLT in the BM-MNC product was reported to inversely correlate with LVEF recovery in patients after cell therapy, even though this negative effect of PLT was less pronounced than that of contaminant RBC; on the contrary, contaminant neutrophils had no significant impact [25]. Regardless of the therapeutic effect, BM-MNC purity is an important parameter with respect to the interpretation of the results of clinical trials. Increasing the product purity has been also specifically requested by regulatory authorities. The majority of development experiments were performed using sternal BM samples, harvested from patients undergoing cardiac surgery involving sternotomy. Such samples, showing a close similarity to iliac crest samples with respect to most analytical parameters, were more easily available in our clinical context with respect to iliac crest BM.

The previous manufacturing protocol used in the initial phase of the METHOD study [11] and in the 'swiss multicenter intracoronary stem cells study in acute myocardial infarction clinical trial" (SWISS-AMI) [12],[13] was derived from prior studies [26] with minor modifications. The new BM-MNC manufacturing protocol relies on the use of a low-density variant of Ficoll-Paque, i. e. Ficoll-Paque PREMIUM 1.073, which was selected based on previous evidence that BM-MNC isolated by Ficoll-Paque with different densities may differ in composition [27]. In agreement with Grisendi et al. [27], we observed that low-density Ficoll-Paque leads to cells with higher mesenchymal stem cell clonogenic potential compared to standard Ficoll-Paque (Figure 1C). However, those authors reported a similar recovery of total nucleated cells for the two media, whereas in our hands the MNC yield was significantly reduced using the new protocol (Table 4), presumably due to the effect of low-density Ficoll-Paque (Table 2). Nevertheless, this limitation is offset by several advantages including higher purity (Table 5) and higher content of hematopoietic and mesenchymal precursors in cell products manufactured with the new method (Figure 1).

Unlike Grisendi et al. [27], we evaluated additional methodological aspects besides the gradient medium. Gradient centrifugation parameters were adjusted (from 719 × g, 20' at 20°C to 400 × g, 30' at 20°C) to better adhere to the Ficoll-Paque manufacturer's instructions; such adjustment itself had no impact on overall process performance (Table 1).

The washing procedure was also modified (from three washing steps at 582 × g, 10' at 4°C, to two washing steps at 400 × g and 100 × g, respectively, both at 20°C), on the basis of Ficoll-Paque manufacturer's indications suggesting two centrifugations, one of which may be performed at low speed to improve PLT removal. In our hands, the use of a low-speed centrifugation step indeed contributed to PLT abatement, even though it further reduced MNC yield (Table 3). The filtration steps were not optimized as they do not affect process performance (data not shown). The purpose of the initial filtration (100 μm) is to remove cell clumps or BM tissue fragments that could interfere with subsequent process steps, while the goal of the final filtration (70 μm) is to withdraw possible cell aggregates from the final product. Overall, the new method significantly ameliorated product quality.

With respect to the QC strategy applied in most previous BM-MNC cardiovascular cell therapy studies [2],[28],[29], our approach is more stringent in terms of safety, as in those studies cells were tested for sterility/microbiological contamination [30]-[34] but not for endotoxin. Endotoxin testing is important because high endotoxin levels as a result of bacterial contamination and/or improper quality of manufacturing materials and reagents cannot be ruled out a priori. Moreover, this is the only safety test which yields results before fresh product infusion, since microbiological assays take 7 days or longer. The specification for endotoxin (<5 Endotoxin Units/mL) has been set according to EP [35].

Cell viability was evaluated in most trials [26],[30],[32],[34],[36]-[44] and defined release criteria were reported by some authors, e.g. > 70% viable cells in the FOCUS-CCTRN trial [30],[31] and > 80% in the REPAIR-AMI trial [31]. In our case, the specification was set at ≥ 70% in accordance to recent FDA guidelines [21].

Cell immunophenotype (mainly CD45 and CD34) was analyzed in most previous studies [30]-[34],[36],[39],[41]-[44], even though no release specifications were reported. We extended the immunophenotypic analysis to include not only CD133, a stem cell marker expressed by a subset of CD34⁺ cells [19],[45] but also CD184/CXCR4 (SDF-1 receptor), a marker of functional BM-MNC activity. Seeger et al. [20] reported that injection of CXCR4⁺ BM-MNC in mice with hindlimb ischemia significantly improved the recovery of perfusion compared to injection of CXCR4-negative BM-MNC; likewise, capillary density was significantly increased in mice treated with CXCR4⁺ BM-MNC. The higher migratory capacity of such cells and the release of paracrine factors may have contributed to tissue repair [20]. A correlation between in vitro cell potency and long term clinical outcome has been recently shown for BM-MNC therapy of acute myocardial infarction [46].

In the present study, the presence of CD34⁺ CD133⁺ CD309⁺ EPC [47] was evaluated on representative BM-MNC batches (Table 7, n = 5); however, it is not part of the QC release panel due to the limited number of available cells in clinical batches and the relatively high cell number required for this analysis.

In terms of functional cell characterization, we included in QC release panel colony-forming assays for both hematopoietic (CFC) and mesenchymal precursors (CFU-F), along with testing of invasion capacity, which were assessed in the REPAIR-AMI study [25] and, in part, in the PROVASA study [39]. In addition, we performed the CFU-EC assay to detect "angiogenic cells" [48].

In previous studies, limited data on cell characteristics may have hampered the assessment of critical cell product parameters. The poor product characterization also limited the evaluation of product comparability among cardiovascular cell therapy studies, thus contributing to variable clinical results [28]. It is well known that BM-MNC isolated through density gradient consist of a mixture of various cell subsets with different phenotype and function [49],[50], including progenitor/stem cells such as hematopoietic stem cells, mesenchymal stem cells, EPC, multipotent adult mesenchymal progenitors and embryonic-like stem cells. The cell subsets responsible for beneficial effects of total BM-MNC in several clinical studies are not yet identified [20],[51]. It is also unknown whether selected BM-derived cell subpopulations may be superior to unfractionated BM populations containing a mixture of differentiated and less differentiated cells, potentially enhancing their effect by cross-talking with each other [51].

Based on these considerations, our immunophenotypic and functional analysis of clinical grade BM-MNC may well contribute to the identification of product characteristics having impact on the clinical outcome in different patient populations, thus facilitating the identification of product critical quality attributes and the definition of release specifications for further clinical studies [52].

The last part of our work focused on product stability, an issue particularly relevant in the case when a freshly-derived cell product is delivered to the patient. Due to the peculiar nature of the product (live cells, intrinsic inter-batch variability due to differences among patients, limited product availability), the stability study was performed at the storage temperature only (10 ± 5°C), as accelerated and stressed conditions recommended in general stability guidelines [53],[54] were considered not applicable for viable cells. Both release parameters and characterization parameters not yet subjected to release specifications were evaluated within the context of stability testing (Figure 2). Overall, the results suggested that the product shelf-life could be set at 20 hours. This time frame abundantly covers the time necessary for the release of the product: pre-infusion testing (endotoxin, cell concentration, cell viability, immunophenotype) generally requires approximately 2 hours, or 4 hours in case of retesting due to deviations or non-conformities; the remaining 16 hours may cover the transport to the clinical site and/or infusion delay due to clinical issues.

Principles detailed in Swiss and European regulations for ATMP [4]-[6], as well as in the applicable European Medicinal Agency guidelines [7] and GMP guidelines [55], were taken into consideration throughout our development work. This approach allowed us to fulfil requests formulated by the competent regulatory authorities in view of the upcoming second phase of the METHOD trial [11] and of the new CIRCULATE study. Based on the results summarized here, included in the quality section of Investigational Medicinal Product Dossier, the CIRCULATE clinical trial was successfully submitted and recently got authorization.

Methods for BM-MNC production and testing have been optimized and validated according to GMP. In particular, the manufacturing process has been redesigned, resulting in higher product purity and activity. Additional identity and potency assays have been set up in order to extend product characterization and evaluate product stability: an extended QC panel has been established, encompassing safety, identity/purity, and potency. Release specifications have been updated and product shelf-life has been defined based on experimental results obtained during development and GMP validation. The present work represents an example of constructive cooperation between a cell therapy manufacturing site and regulatory authorities, whose valuable inputs have been considered during product development.

MR conceived and designed the study, carried out experiments, acquired/analyzed/interpreted data, performed statistical analysis, and drafted the manuscript; VLC carried out experiments, acquired/analyzed/interpreted process and QC data, and revised the manuscript; SS and SB carried out experiments and acquired/analyzed/interpreted QC data; DS provided study material, discussed data, and revised the manuscript; TT provided study material; FS provided study material; TM discussed data and revised the manuscript; GV discussed data and revised the manuscript; LT conceived and designed the study, carried out experiments, acquired/analyzed/interpreted data, and revised the manuscript. All authors read and approved the final manuscript.